Registration Dossier

Administrative data

Endpoint:
toxicity to terrestrial plants: short-term
Type of information:
experimental study
Adequacy of study:
key study
Study period:
5th November 2008 to 6th April 2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report Date:
2009

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 208 (Terrestrial Plants Test: Seedling Emergence and Seedling Growth Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Sponsor's identification: Experimental additive 20735-35

The integrity of supplied data relating to the identity, purity and stability of the test material is the responsibility of the Sponsor.

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
The concentration of the test material in the soil was verified by chemical analysis on Day 0 for each preparation.

- Concentrations: Duplicate samples were taken
- Sampling method: Not specified
- Sample storage conditions before analysis: stored at approximately -20°C

Test substrate

Vehicle:
no
Details on preparation and application of test substrate:
- Method of application to filter paper (if used): Not applicable
- Method of mixing into soil (if used): For the purpose of the definitive test the test material was added directly to the soil.
An amount of test material (10.5 g) was mixed with approximately 100 g of sand prior to incorporation into 10.5 kg (dry weight) of soil using a Hobart A200N mixer to give the 1000 mg/kg test concentration.
- Method of application to soil surface (if used): Not applicable
- Controls: The control was soil substrate mixed with approximately 100 g of sand without the addition of test material.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): Not applicable
- Concentration of vehicle in test medium (stock solution and final test solution): Not applicable
- Evaporation of vehicle before use: Not applicable

Test organisms

open allclose all
Species:
Glycine max (G. soja)
Plant group:
Dicotyledonae (dicots)
Details on test organisms:
- Common name: Soybean
- Plant family: Dicotyledonous species
- Variety: Amsoy
- Source of seed: Herbiseed, New Farm, Mire Lane, West End, Twyford, UK
- Prior seed treatment/sterilization: The seeds were supplied untreated.
- Historical germination of seed (germination of seed lot tested): Range finding test - percentage germination (supplied by seed supplier): 91%; Definitive test - percentage germination (supplied by seed supplier): 82%
- Seed storage: Upon arrival the seeds were stored under cool, dry conditions.
Species:
Lycopersicon esculentum
Plant group:
Dicotyledonae (dicots)
Details on test organisms:
- Common name: Tomato
- Plant family: Dicotyledonous species
- Variety: Orange Pixie
- Source of seed: Herbiseed, New Farm, Mire Lane, West End, Twyford, UK
- Prior seed treatment/sterilization: The seeds were supplied untreated.
- Historical germination of seed (germination of seed lot tested): Range finding test - percentage germination (supplied by seed supplier): 89%; Definitive test - percentage germination (supplied by seed supplier): 89%
- Seed storage: Upon arrival the seeds were stored under cool, dry conditions.
Species:
Avena sativa
Plant group:
Monocotyledonae (monocots)
Details on test organisms:
- Common name: Oat
- Plant family: Monocotyledonous species
- Variety: Banquo
- Source of seed: Senova Ltd., 49 North Road, Great Abington, Cambridge, UK
- Prior seed treatment/sterilization: The seeds were supplied untreated.
- Historical germination of seed (germination of seed lot tested): Range finding test - percentage germination (supplied by seed supplier): 97%; Definitive test - percentage germination (supplied by seed supplier): 90%
- Seed storage: Upon arrival the seeds were stored under cool, dry conditions.

Study design

Test type:
other: Seedling emergence and seedling growth
Study type:
laboratory study
Substrate type:
artificial soil
Limit test:
yes
Total exposure duration:
21 d
Post exposure observation period:
Not performed

Test conditions

Test temperature:
10-47 °C
pH:
The initial pH of the soil was 8.3. However, pH measurements were not performed throughout the test.
Moisture:
After the initial watering the pots were watered as necessary using sub-irrigation.
Details on test conditions:
TEST SYSTEM
- Testing facility: Laboratory greenhouse
- Test container (type, material, size): The exposure vessels consisted of polypropylene pots with a depth of 9.9 cm and a diameter of 13 cm.
- Amount of soil: Each pot contained approximately 850 g (dry weight) of substrate.
- Method of seeding: Not specified
- No. of seeds per container: 5 seeds/ container
- No. of plants (retained after thinning): Thinning was not performed
- No. of replicates per treatment group: 4 replicates/ species
- No. of replicates per control: 4 replicates
- No. of replicates per vehicle control: Not applicable

SOURCE AND PROPERTIES OF SUBSTRATE (if soil)
- Composition (if artificial substrate): The soil was supplied by Sporting Surface Supplies Ltd, Smallfield, Surrey, UK. The soil was characterised as a sand with 0.8% organic carbon content and a pH of 8.3.
- Organic carbon (%): 0.8%
- Maximum water holding capacity (in % dry weigth): Not specified
- CEC: Not specified
- Pretreatment of soil: Not specified
- Storage (condition, duration): Not specified

NUTRIENT MEDIUM (if used)
- Description: Not applicable

GROWTH CONDITIONS
- Photoperiod: Approximately 16 hours light and 8 hours darkness
- Light source: Artificial lighting
- Light intensity and quality: Not specified
- Day/night temperatures: Not specified
- Relative humidity (%): 14-69%
- Wind velocity: Not specified
- Air exchange rate: Not specified
- Watering regime and schedules:
- Water source/type: Not specified
- Volume applied: Not specified - water was provided as necessary.
- Interval of applications: Water was provided as necessary
- Method of application: After planting, each pot was placed in a plant saucer which served as a sub-irrigation reservoir. Initial watering was via the surface to encourage germination. All subsequent watering was by sub-irrigation as required.
- Any pest control method/fertilization (if used): None specified

ACCLIMATION PERIOD: Not specified

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): All the pots were monitored daily after sowing for emergence and the date when 50% emergence occurred in the control pots for each species was recorded. For 21 days following 50% emergence in the control pots, the number of seedlings emerged and any mortalities and/or morphological abnormalities were determined daily in the test and control pots for each species. On Day 21 the above ground portion of each seedling was harvested and the dry weight determined by placing in pre-weighed containers and drying in an oven at approximately 60°C to a constant weight.

- Phytotoxicity rating system (if used): None specified

VEHICLE CONTROL PERFORMED: No - not applicable

TEST CONCENTRATIONS
- Spacing factor for test concentrations: Not applicable - a limit test was performed.
- Justification for using less concentrations than requested by guideline: Based on the results of the range-finding test a "Limit test" was conducted at a concentration of 1000 mg/kg using the test species soybean (Glycine max), tomato (Lycopersicon esculentum) and oat (Avena sativa) to confirm that at the maximum concentration given in the OECD Test Guidelines no effect on seedling emergence or morphological abnormalities were observed.
- Range finding study
- Test concentrations: Pre-study solubility work indicated that the test material was insoluble in water and recommended auxiliary solvents. Therefore, it was considered appropriate to mix the test material directly into the soil as recommended in the test guideline for poorly soluble test materials.
Amounts of test material (51, 510 and 5100 mg) were each separately mixed with approximately 100 g of sand prior to incorporation into 5.1 kg (dry weight) of soil using a Hobart A200N mixer to give the 10, 100 and 1000 mg/kg test concentrations respectively.
In the range-finding test, five seeds were placed in each test and control pot for each species. Two replicates were prepared for each concentration. Following sowing, the pots were surface irrigated to encourage germination and then placed in a randomised fashion in the greenhouse. The temperature was maintained at 1°C to 32°C with a photoperiod of approximately 16 hours light and 8 hours darkness. Some of the temperatures were observed to be outside the range given in the protocol. This deviation was considered not to affect the integrity or the outcome of the study as no morphological abnormalities were observed in the control pots. After the initial watering the pots were watered as necessary using sub-irrigation.
The pots were monitored daily after sowing for emergence and the date when 50% emergence occurred in the control pots for each species was recorded. For 21 days following 50% emergence in the control pots the number of seedlings emerged and any mortalities and/or morphological abnormalities were determined daily in the test and control pots for each species. On Day 21 the above ground portion of each seedling was harvested and the dry weight determined by placing in pre-weighed containers and drying in an oven at approximately 60°C to a constant weight.
- Results used to determine the conditions for the definitive study: A summary of the morphological effects and mortalities for each species during the range-finding test is given in Table 1.
There were no significant differences (P>=0.05) between the control and all the test groups in terms of emergence at test termination for each species.
There were no significant decreases in shoot dry weight (P>=0.05) between the control and all the test groups at test termination for each species.
Based on this information, a single test concentration of four replicates, of 1000 mg/kg was selected for each species for the definitive test. This experimental design conforms to a "Limit test" to confirm that at the maximum test concentration given in the OECD/EEC Test Guidelines no effect on seedling emergence or morphological abnormalities were observed.
Nominal and measured concentrations:
In the range-finding test soybean (Glycine max), tomato (Lycopersicon esculentum) and oat (Avena sativa) were exposed to a series of nominal test concentrations of 10, 100 and 1000 mg/kg.
Based on the results of the range-finding test a "Limit test" was conducted at a concentration of 1000 mg/kg using the test species soybean (Glycine max), tomato (Lycopersicon esculentum) and oat (Avena sativa) to confirm that at the maximum concentration given in the OECD Test Guidelines no effect on seedling emergence or morphological abnormalities were observed.
Reference substance (positive control):
no

Results and discussion

Effect concentrationsopen allclose all
Species:
Glycine max (G. soja)
Duration:
21 d
Dose descriptor:
EC50
Effect conc.:
> 1 000 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
seedling emergence
Species:
Glycine max (G. soja)
Duration:
21 d
Dose descriptor:
EC50
Effect conc.:
> 1 000 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth
Species:
Glycine max (G. soja)
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
1 000 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: seedling emergence and growth
Species:
Lycopersicon esculentum
Duration:
21 d
Dose descriptor:
EC50
Effect conc.:
> 1 000 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
seedling emergence
Species:
Lycopersicon esculentum
Duration:
21 d
Dose descriptor:
EC50
Effect conc.:
> 1 000 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth
Species:
Lycopersicon esculentum
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
1 000 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: seedling emergence and growth
Species:
Avena sativa
Duration:
21 d
Dose descriptor:
EC50
Effect conc.:
> 1 000 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
seedling emergence
Species:
Avena sativa
Duration:
21 d
Dose descriptor:
EC50
Effect conc.:
> 1 000 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth
Species:
Avena sativa
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
1 000 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: seedling emergence and growth
Details on results:
SEEDLING EMERGENCE
- Percent seedling emergence: Soybean (Glycine max), tomato (Lycopersicon esculentum) and oat (Avena sativa) reached 50% emergence in the control on Days 7, 15 and 6 respectively after sowing.
A summary of the percentage emergence for each species are given in Table 2.
There were no significant differences (P>=0.05) between the control and 1000 mg/kg test group in terms of emergence at test termination for all three species.
- Percent survival: A single mortality was observed in the tomato at 1000 mg/kg on Day 13 of the definitive test. This was considered to be due to natural causes rather than a toxic effect given that no further mortalities or phytotoxic effects were observed.
- Plant height: Not specified
- Dry weight: Shoot mean dry weight values for each species are given in Table 4.
There were no significant differences (P>=0.05) between the control and 1000 mg/kg test group in terms of emergence at test termination for all three species.
- Abnormal seed development or appearance: A summary of any morphological abnormalities (for example, chlorosis or necrosis of the leaves) and mortalities, for each species is given in Table 3.
No morphological abnormalities were observed for each species throughout the test
- Other effects: No other effects were reported.
Results with reference substance (positive control):
Not applicable
Reported statistics and error estimates:
95% confidence limits could not be determined for the test material.

Any other information on results incl. tables

Analysis of the 1000 mg/kg test sample from the initial preparation (tomato and oat) showed a measured concentration of 135% of nominal with 121 mg/kg detected in the control sample. Analysis of the frozen duplicate samples showed measured concentrations of less than the limit of quantitation to 63% of nominal for the control and 1000 mg/kg test sample respectively. It was therefore considered that the initial values were due to post sampling contamination. Analysis of the 1000 mg/kg test sample from the second preparation (soybean) showed a measured concentration of 72% of nominal. Although the levels detected were slightly below the 80% acceptance limit it was considered that the test system had been correctly dosed and that the slightly low measured concentrations were possible due to the test material adhering to the test soil and so the results are based on nominal test concentrations only.

Table 1: Observations of morphological effects and mortalities in the range-finding test

Species

Nominal concentration (mg/kg)

Morphological effects* (population = 5 seeds per pot, 2 replicates per control and test concentration)

Mortality**

Chlorosis

Necrosis

Soybean

(Glycine max)

Control

0

0

2

10

0

0

1

100

0

0

0

1000

0

0

0

Tomato

(Lycopersicon esculentum)

Control

0

0

3

10

0

0

1

100

0

0

4

1000

0

0

0

Oat

(Avena sativa)

Control

0

0

1

10

0

0

0

100

0

0

0

1000

0

0

1

* number of plants exhibiting the effect

** number of non-emerged plants

Table 2: Percentage seedling emergence in the definitive test

Crop

Concentration (mg/kg)

Mean % emergence

Standard deviation

Soybean

(Glycine max)

Control

85

19

1000

90

12

Tomato

(Lycopersicon esculentum)

Control

100

0

1000

100

0

Oat

(Avena sativa)

Control

85

19

1000

95

10

Table 3: Observations of morphological effects and mortalities in the definitive test

Species

Nominal concentration (mg/kg)

Morphological abnormalities*

Mortality**

Chlorosis

Necrosis

Soybean

(Glycine max)

Control

0

0

3

1000

0

0

2

Tomato

(Lycopersicon esculentum)

Control

0

0

0

1000

0

0

1***

Oat

(Avena sativa)

Control

0

0

3

1000

0

0

1

* number of plants exhibiting the effect

** number of non-emerged plants from a total of 20 (4 replicates of 5 seeds)

*** single mortality observed on day 13

Table 4: Mean shoot dry weights

Crop

Concentration (mg/kg)

Mean shoot dry weight (g)

Standard deviation

Soybean

(Glycine max)

Control

0.3853

0.0594

1000

0.3232

0.1254

Tomato

(Lycopersicon esculentum)

Control

0.0972

0.1029

1000

0.0802

0.0505

Oat

(Avena sativa)

Control

0.0934

0.0213

1000

0.0906

0.0223

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
The test material, Millad 3988, had no effect on the seedling emergence and growth of three plant species, soybean (Glycine max), tomato (Lycopersicon esculentum) and oat (Avena sativa). The EC50 values for both growth and emergence for all three species were >1000 mg/kg and the NOEC was 1000 mg/kg.
Executive summary:

A study was performed to assess the effects of the test material on the emergence and growth of three plant species. The method followed that described in the OECD Guidelines for Testing of Chemicals: Guideline 208: Terrestrial (Non-target) Plant Test: Seedling Emergence and Seedling Growth Test (July 2006).

The results of the study demonstrated that the test material, Millad 3988, had no effect on the seedling emergence and growth of three plant species, soybean (Glycine max), tomato (Lycopersicon esculentum) and oat (Avena sativa). The EC50 values for both growth and emergence for all three species were >1000 mg/kg and the NOEC was 1000 mg/kg.